Methylotrophic yeasts are those yeasts that are able to utilize methanol as a sole source of carbon and energy. Species of yeasts that have the biochemical pathways necessary for methanol utilization are classified in four genera, Hansenula, Pichia, Candida, and Torulopsis. These genera are somewhat artificial, having been based on cell morphology and growth characteristics, and do not reflect close genetic relationships (Billon-Grand, Mycotaxon 35:201-204, 1989; Kurtzman, Mycologia 84:72-76, 1992). Furthermore, not all species within these genera are capable of utilizing methanol as a source of carbon and energy. As a consequence of this classification, there are great differences in physiology and metabolism between individual species of a genus.
Methylotrophic yeasts are attractive candidates for use in recombinant protein production systems. Some methylotrophic yeasts have been shown to grow rapidly to high biomass on minimal defined media. Certain genes of methylotrophic yeasts are tightly regulated and highly expressed under induced or de-repressed conditions, suggesting that promoters of these genes might be useful for producing polypeptides of commercial value. See, for example, Faber et al., Yeast 11:1331, 1995; Romanos et al., Yeast 8:423, 1992; and Cregg et al., Bio/Technology 11:905, 1993.
Development of methylotrophic yeasts as hosts for use in recombinant protein production systems has been slow, due in part to a lack of suitable materials (e.g., promoters, selectable markers, and mutant host cells) and methods (e.g., transformation techniques). The most highly developed methylotrophic host systems utilize Pichia pastoris and Hansenula polymorpha (Faber et al., Curr. Genet. 25:305-310, 1994; Cregg et al., ibid.; Romanos et al., ibid.; U.S. Pat. No. 4,855,242; U.S. Pat. No. 4,857,467; U.S. Pat. No. 4,879,231; and U.S. Pat. No. 4,929,555).
More recently, materials and techniques useful for producing foreign proteins in Pichia methanolica have been developed (WIPO Publication WO 9717450). However, there remains a need in the art for additional techniques that can be used to manipulate the genome of P. methanolica so as to expand our understanding of this organism and produce strains that can be used in large-scale protein production systems.
One such needed tool is a technique for directed mutagenesis of P. methanolica. Directed mutagenesis allows the introduction of mutations into predetermined genomic loci, permitting the selective alteration of gene activity. Useful alterations include, for example, mutation of promoter sequences to increase gene expression, introduction of heterologous genes at particular sites, and generation of protease deficiencies and auxotrophies. Techniques developed for the budding yeast Saccharomyces cerevisiae are unsuitable for P. methanolica. For example, the "pop-in/pop-out" method developed by Scherer and Davis (Proc. Natl. Acad. Sci. USA 76:1035, 1979) and summarized by Rothstein (Methods Enzymol. 194:281, 1991) requires a selection against the presence of the URA3 marker, such as by addition of 5-FOA (5 fluoro orotic acid) to the culture medium. This method is unsuitable with P. methanolica because the cells are resistant to 5 fluoro-orotic acid (5-FOA), and no P. methanolica URA marker is available. The present invention provides methods for producing directed mutations in the genome of P. methanolica, cells having such mutations, and other, related advantages.